Bacterial vaginosis (BV) results from a disruption of the vaginal ecosystem characterized by a complex shift in the microflora. Concentrations of the normally dominant Lactobacillus decrease while other microflora increase (Prevotella, Peptostreptococcus, Gardnerella, Mobiluncus). Studies have linked BV with upper genital tract infections and adverse pregnancy outcomes, particularly preterm delivery (PTD). Bacteria can weaken fetal membranes through production of soluble factors that lead to PTD either by induction of a proinflammatory response or stimulation of prostaglandin E2 production. An inflammatory response leading to PTD can also be stimulated in host cells directly through attachment and internalization of bacteria. Our laboratory has developed in vitro models of the vaginal ecosystem that combine mixed cultures of normal (NMVF) and BV-associated (BVAF) bacteria with immortalized cervical and vaginal epithelial cells in coculture. Preliminary studies demonstrate these are viable models for studying bacterial-epithelial interactions of the vaginal ecosystem. Data from studies using NMVF and both cell lines indicate that all microflora components adhere to the epithelial cells, but only Lactobacillus (La), Prevotella (Pb), and Enterococcus are internalized. In addition, Pb stimulated interleukin-8 production while La induced significant apoptosis in cocultures. This application is divided into 2 aims designed to analyze the role of BV-associated microflora (BVAF) in the pathogenesis of PTD using these unique models. The inflammatory response to coculture will be characterized, comparing findings using NMVF and BVAF. Adherence, internalization, and apoptosis rates will be determined for BVAF and NMVF and compared. Bacterial factors produced during coculture will be determined and correlated with stimulation of an inflammatory response. Due to the novelty of the models, we have the unique opportunity to determine the role of BVAF in PTD.